We have employed confocal microscopy to determine the in situ localization of GLUT4 in rat adipose cells. We find that the basal compartment from which GLUT4 is translocated in response to insulin comprises specialized post-endosamal VAMP2-positive vesicles, distinct from the constitutively recycling endosomes. These observations are consistent with a kinetic model in which GLUT4 is sequestered through two or more intracellular pools in series. Vp165, an aminopeptidase, is one of the major proteins of the GLUT4-containing vesicles. We have determined the degree of co-localization between vp165 and GLUT4 in rat adipose cells and used perturbation by wortmannin to assess the exocytic and endocytic steps along the translocation and recycling pathways of GLUT4. The results are consistent with the presence of a distinct insulin-sensitive compartment that sequesters both GLUT4 and vp165 and suggest similar trafficking routes through the recycling compartments. We have examined the subcellular distribution of a member of the Rab 3 family, Rab 3D, in rat adipose cells. We find that if Rab 3D were to play a role in GLUT4 trafficking, it must rely on mechanisms independent of relocation. Nevertheless, we observe that Rab 3D is overexpressed in adipose cells of obese (fa/fa) Zucker rats, thus representing a potential molecular basis for altered adipose secretory function in obesity. The effects of exercise training on maximal glucose transport activity and cell surface GLUT4 have been examined in rat epitrochlearis muscle. The results demonstrate that changes in insulin- and hypoxia-stimulated glucose transport activity after exercise training are fully accounted for by the appearance of cell surface GLUT4 and support the concept of two intracellular pools of GLUT4. The effects of exercise training on cell surface GLUT4 in skeletal muscle of the obese (fa/fa) Zucker fatty rat have been investigated using the impermeant glucose transporter photoaffinity reagent ATB-BMPA. The results indicate that training improves insulin-stimulated glucose transport in muscle of the obese Zucker rat by increasing GLUT4 content and by altering the normal intracellular distribution of these transporters such that they are now capable of migrating to the cell surface in response to the insulin stimulus. We have also assessed cell surface GLUT4 and measured the corresponding glucose transport activity in isolated extensor digitorum longus (EDL) muscles from non-transgenic (NTG) and GLUT4 TG mice. The results suggest that: 1) alterations in glucose transport activity which occur with GLUT4 overexpression in EDL muscles are directly related to cell surface GLUT4 content, 2) while overexpression of GLUT4 influences both basal and insulin-stimulated glucose transport activity, the glucose transport response to hypoxia/contraction is unchanged, and 3) subcellular fractionation provides little insight into the subcellular trafficking of GLUT4, and whatever relationship is demonstrated in EDL muscles from NTG mice is disrupted on GLUT4 overexpression.